EP2940136A1 - Method for isolating poly(A) nucleic acids - Google Patents

Method for isolating poly(A) nucleic acids Download PDF

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Publication number
EP2940136A1
EP2940136A1 EP14166712.1A EP14166712A EP2940136A1 EP 2940136 A1 EP2940136 A1 EP 2940136A1 EP 14166712 A EP14166712 A EP 14166712A EP 2940136 A1 EP2940136 A1 EP 2940136A1
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European Patent Office
Prior art keywords
poly
hybridization
nucleic acids
hybridization solution
nucleic acid
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EP14166712.1A
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German (de)
English (en)
French (fr)
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Gabriele Christoffel
Martin Schlumpberger
Dominic O'neil
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Qiagen GmbH
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Qiagen GmbH
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Application filed by Qiagen GmbH filed Critical Qiagen GmbH
Priority to EP14166712.1A priority Critical patent/EP2940136A1/en
Priority to CN201580023578.1A priority patent/CN106459965A/zh
Priority to CN202111546872.4A priority patent/CN114395552A/zh
Priority to PCT/EP2015/059117 priority patent/WO2015165859A1/en
Priority to US15/129,280 priority patent/US20180179514A1/en
Priority to EP15722465.0A priority patent/EP3137600B1/en
Publication of EP2940136A1 publication Critical patent/EP2940136A1/en
Priority to US17/344,792 priority patent/US20210380966A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads

Definitions

  • the present invention provides a method for isolating poly(A) nucleic acids from a nucleic acids containing sample.
  • the described method efficiently enriches poly(A) nucleic acids such as poly(A) RNA while depleting unwanted non-poly(A) nucleic acids such as e.g. rRNA.
  • the method is particularly suitable for preparing poly(A) RNA for next generation sequencing (NGS) applications.
  • NGS next generation sequencing
  • compositions and kits suitable for performing the method according to the present invention are provided.
  • polyadenylation commonly refers to the addition of a stretch consisting of multiple (usually tens to hundreds) of contiguous adenine (A) residues to a biomolecule, wherein the stretch is usually present at the 3' end of the molecule and commonly is referred to as "poly(A) tail".
  • Nucleic acids containing a respective poly(A) stretch or poly(A) tail are commonly referred to as "poly(A) nucleic acids”.
  • polyadenylation is a process associated with the production of mature messenger RNA (mRNA) for gene expression (translation).
  • RNA Ribonuclear polyadenylation
  • RNA 14 (1): 1-10, 2007 animal replication-dependent histone mRNAs
  • RNA 14 (1): 1-10, 2007 animal replication-dependent histone mRNAs
  • various eukaryotic non-coding RNAs are polyadenylated. This also includes some small RNAs, such as e.g. microRNAs which may have a poly(A) tail in their intermediary forms during microRNA maturation.
  • nucleic acids can also be artificially polyadenylated to provide them with a poly(A) tail.
  • the poly(A) tail of nucleic acids has been used as a means for isolating poly(A) nucleic acids from different sample types and in particular has been used for separating poly(A) nucleic acids from nucleic acids that lack a poly(A) tail, also referred to herein as "non-poly(A) nucleic acids".
  • Poly(A) nucleic acids can be isolated with the aid of a probe capable of hybridizing to the single-stranded poly(A) stretch.
  • probes can capture the poly(A) nucleic acid by hybridization to the poly(A) stretch and hereinafter are also referred to as capture probe.
  • an oligonucleotide comprising a sequence complementary to the poly(A) stretch of the poly(A) nucleic acids is used for that purpose, herein also referred to as capture oligonucleotide.
  • capture oligonucleotide comprising a sequence complementary to the poly(A) stretch of the poly(A) nucleic acids is used for that purpose, herein also referred to as capture oligonucleotide.
  • a solid support is used that is functionalized with the capture probe.
  • oligo dT complementary DNA oligonucleotides
  • the column is then subjected to extensive washing with the application buffer (containing 0.5 M KCI), then a lower-ionic-strength solution (0.1 M KCI), followed by elution of mRNA with 10 mM Tris (pH 7.5).
  • the hybridization can be performed in solution, linking the oligo-dT-mRNA hybrids to the solid support in a subsequent step.
  • This procedure has tended to be an inefficient method for selectively isolating poly(A) RNA while depleting rRNA. rRNA carryover levels are often high enough to provide the same problems as presented with total RNA, especially for analysis of rare transcripts.
  • Isostabilizing agents such as quaternary ammonium salts belonging to the group of tetramethylammonium (TMA+) and tetraethylammonium (TEA+) ions and the glycine amino acid derivative betaine, equalize the hydrogen bonding strength of the A:T and G:C base pairs when used at the appropriate concentrations (Jacobs et al., 1988; Jacobs et al., 1985; Gitschier et al., 1986; Melchior et al., 1973; Rees et al., 1993; Wood et al., 1985; WoZney, 1990).
  • TMA+ tetramethylammonium
  • TEA+ tetraethylammonium
  • Such isostabilizing agents were used in the art to facilitate poly(A) isolation.
  • US 6,812,341 describes a poly(A) enrichment method which aims at reducing rRNA carry-over during the isolation process by using isostabilizing agents such as tetramethlyammonium (TMA+) and tetraethylammonium (TEA+) ions, preferably TMAC or TEAC.
  • WO 90/12116 relates to a generic method wherein oligo-dT -coated magnetic particles are used for the isolation of poly(A) RNA.
  • tetraalkylammonium cations are used to stabilize the A:T bonds between the poly(A) tail of the poly(A) nucleic acid and the oligo(dT) probes in combination with chaotropic salts.
  • poly(A) nucleic acid isolation such as the MagAttract® Direct mRNA M48 kit which uses oligo (dt) capture oligonucleotides immobilized on magnetic beads or the Oligotex® mRNA Kit for isolation of poly(A) RNA (Qiagen) which uses an oligo (dT) capture oligonucleotides immobilized to polystyrene-latex beads as solid support.
  • MagAttract® Direct mRNA M48 kit which uses oligo (dt) capture oligonucleotides immobilized on magnetic beads
  • Oligotex® mRNA Kit for isolation of poly(A) RNA (Qiagen) which uses an oligo (dT) capture oligonucleotides immobilized to polystyrene-latex beads as solid support.
  • NGS next generation sequencing
  • RNA-seq RNA-sequencing
  • RNA species which do not carry a poly(A) tail such as rRNA (which is not of interest) are in theory not recovered and are accordingly not carried over into the sequencing reaction.
  • rRNA ribosomal RNA
  • transcriptome sequencing is the presence of interfering RNA molecules in particular if the poly(A) RNA starting material is not of sufficient purity and contains non-poly(A) contaminations. If abundant rRNA is involved in library construction, sequencing power will be used to sequence these ubiquitous molecules. The highly abundance of rRNA may predominate in the sequencing reads, thereby hindering the study of lowly expressed genes and wasting valuable sequencing resources. Furthermore, the presence of ribosomal RNA may result in a low signal-to-noise ratio that can make detection of the RNA species of interest difficult. Therefore, improving the depletion of rRNAs and/or other unwanted non-poly(A) RNA during isolation of the poly(A) RNA increases the value of the downstream sequencing because more information can be deduced from a sequencing run.
  • poly(A) nucleic acid enrichment usually requires two or more rounds of poly(A) nucleic acid enrichment to provide poly(A) nucleic acid samples wherein the amount of non-poly(A) nucleic acid such as e.g. rRNA is sufficiently low to provide a useful sample for certain applications such as NGS applications where the purity requirements are high.
  • This requirement for repetitions of the entire poly(A) nucleic acid isolation procedure can lead to several disadvantageous side effects and furthermore, extends the hours required to perform the procedure.
  • the representational distribution of various poly(A) nucleic acids may become altered or more altered, or the unavoidable losses associated with the repeated procedure may reduce the level of the commonly-sought low-abundance messages beyond the limits of detection.
  • one aim is to provide an improved method for isolating poly(A) nucleic acids for next generation sequencing.
  • products suitable for performing respective methods shall be provided.
  • the present invention is inter alia based on the surprising finding that nucleic acids containing a single stranded poly(A) stretch (in the following also referred to as "poly(A) nucleic acids”) can be efficiently and specifically isolated using a capture probe and hybridization conditions which involve the use of a sodium salt in combination with a quaternary ammonium salt. Contaminations with non-poly(A) nucleic acids are reduced when using the hybridisation conditions described herein. As is demonstrated by the examples, the hybridization conditions used in the method according to the invention ensure efficient capture and hence isolation of the poly(A) nucleic acids while significantly reducing binding and hence carry-over of unwanted non-poly(A) nucleic acids into the isolated poly(A) nucleic acids.
  • the invention can be used to specifically and efficiently isolate poly(A) RNA from a nucleic acid containing sample while depleting unwanted non- poly(A) RNA such as rRNA during the isolation process.
  • the method is particularly suitable for preparing poly(A) RNA for next generation sequencing (NGS) applications such as transcriptome sequencing.
  • NGS next generation sequencing
  • the advantageous hybridization conditions which use a sodium salt in combination with a quaternary ammonium salt not only provide stringent and selective capture conditions for poly(A) nucleic acids, but also provide advantageous washing conditions.
  • the hybridization conditions described herein therefore, can be advantageously used to remove non-poly(A) nucleic acids that may have bound to the capture probe and/or the captured poly(A) nucleic acids during washing.
  • a method for isolating poly(A) nucleic acids having a single stranded poly(A) stretch from a nucleic acid containing sample comprising:
  • a method for sequencing poly(A) nucleic acids comprising:
  • This method is particularly suitable for sequencing poly(A) RNA.
  • an aqueous hybridization solution suitable for hybridizing poly(A) nucleic acids to a capture probe capable of hybridizing to the poly(A) stretch of the poly(A) nucleic acids comprising:
  • the hybridization solution according to the third aspect can used in conjunction with and for performing the methods according to the first, second and fourth aspect of the invention. It is particularly suitable for establishing the binding conditions for hybridizing poly(A) nucleic acids to a capture probe while preventing hybridization of non-poly(A) nucleic acids. Furthermore, it can be used to provide stringent washing conditions in order to remove bound non-poly(A) nucleic acids during the washing steps while maintaining hybridization of the poly(A) nucleic acids to the capture probe, thereby increasing the purity of the isolated poly(A) nucleic acid.
  • a kit for isolating poly(A) nucleic acids from a nucleic acid containing sample, comprising:
  • a method for isolating poly(A) nucleic acids having a single stranded poly(A) stretch from a nucleic acid containing sample comprising:
  • the advantageous hybridization solution comprising a sodium salt and a quaternary ammonium salt provided by the present invention is used in the washing step of the isolation process in order to reduce non-poly(A) nucleic acid contaminations during the washing step.
  • the present invention is inter alia based on the surprising finding that poly(A) nucleic acids can be efficiently isolated using a capture probe that can hybridize to the poly(A) stretch (e.g. poly(A) tail) of poly(A) nucleic acids and using hybridization conditions which involve the use of a sodium salt in combination with a quaternary ammonium salt.
  • the hybridization conditions described herein ensure good poly(A) nucleic acid recovery while reducing the amount of unwanted non-poly(A) nucleic acids in the isolated poly(A) nucleic acids.
  • a method for isolating poly(A) nucleic acids having a single stranded poly(A) stretch from a nucleic acid containing sample comprising:
  • a hybridization composition comprising i) a nucleic acid containing sample from which the poly(A) nucleic acids shall be isolated, ii) a hybridization solution comprising a sodium salt and a quaternary ammonium salt and iii) a capture probe capable of hybridizing to the poly(A) stretch of the poly(A) nucleic acid.
  • Said hybridization composition is incubated under conditions that allow the formation of nucleic acid - hybrids between the poly(A) nucleic acids and the capture probe.
  • the nucleic acid containing sample from which the poly(A) nucleic acids are isolated can be any sample from which poly(A) nucleic acids such as poly(A) RNA are commonly isolated. Details with respect to said nucleic acid containing sample and non-limiting common examples are also described below. According to one embodiment, poly(A) RNA is isolated from total RNA.
  • hybridization conditions are explained subsequently, as they are decisive for achieving efficient binding of poly(A) nucleic acids to the capture probe while non-poly(A) nucleic acids cannot effectively bind and thus are efficiently depleted during the isolation process.
  • the composition of the hybridization composition and hence the hybridization conditions will be explained subsequently.
  • Providing the hybridization composition encompasses the addition of a hybridization solution to the sample.
  • the components of the hybridization solution are preferably added as single solution to the sample.
  • the components of the hybridization solution may also be added separately in any order to the sample e.g. using two or more solutions each containing at least one agent such as the sodium salt and/or the quaternary ammonium salt of the hybridization solution to generate the "hybridization solution" in the sense of the present disclosure.
  • Such procedure also renders a "hybridization solution" and therefore, is encompassed by said term.
  • the volume contributed by the capture probe and the solid support is not considered in the determination of the concentration of the subsequently described components in the hybridization composition. I.e. the capture probe and any solid support or other binding agent used to assist the separation (if used) are neglected in the calculation of the concentration of individual components in the hybridization composition.
  • corresponding components such as e.g.
  • sodium chloride or a quaternary ammonium salt that are not added by the hybridization solution but are contained in the nucleic acid containing sample (if contained therein) are according to one embodiment not considered in the calculation of the concentration of the respective components.
  • concentrations and concentration ranges specified below were calculated neglecting the composition of the nucleic acid containing sample which is, as described herein, preferably a purified nucleic acid sample, that may also be diluted.
  • respective components of the nucleic acid containing sample such as the sodium salt and/or the quaternary ammonium salt are considered in the calculation and in this embodiment, the salt concentration in the hybridization composition can be adjusted by the addition of the hybridization solution to yield the final concentration of the sodium salt and of the quaternary ammonium salt (provided by the nucleic acid containing sample and the hybridization solution) in the hybridization composition according to the specifications as given below.
  • the hybridization solution comprises its components in a concentrated form that allows it to be diluted with the nucleic acid containing sample and/or a dilution solution so as to achieve the proper final concentration in the hybridization composition when mixed with the nucleic acid containing sample and/or the dilution solution.
  • a concentrated hybridization solution has the advantage that the user is more flexible with respect to the volume of the nucleic acid containing sample material and that the overall volume to be processed remains lower.
  • the amount of nucleic acid containing sample can be adjusted to a certain volume by adding a dilution solution such as water or other appropriate solvent and said volume is then mixed with a predetermined volume of the hybridization solution to provide the hybridization conditions of the hybridization composition.
  • This procedure has advantages, because the same volume of (diluted) sample material to a predetermined volume of the hybridization solution can be provided simply by adding the appropriate amount of a suitable dilution solution to the nucleic acid containing sample.
  • a suitable dilution solution E.g. 1 volume of (potentially diluted) nucleic acid containing sample can be contacted with 1 to 10, 1 to 5, 1 to 3 or 1 to 2 volumes of hybridization solution, depending on the composition of the hybridization solution.
  • a ratio of 1 volume (potentially diluted) nucleic acid containing sample to 1 volume hybridization solution (1:2 dilution) is particularly preferred and was also used in several examples.
  • the hybridization solution and hence the hybridization composition comprises a sodium salt.
  • the sodium salt promotes binding of the poly(A) nucleic acids to the capture probe. It may be an anorganic or organic sodium salt.
  • the sodium salt is not a chaotropic salt and accordingly, the sodium salt is a non-chaotropic sodium salt.
  • the hybridization solution or hybridization composition does not contain any chaotropic sodium salt.
  • the sodium salt is a sodium halide.
  • the sodium halide is sodium chloride. It was found that sodium chloride is particularly suitable, because it renders advantageous hybridization conditions when used according to the teachings of the present invention.
  • the hybridization composition comprises the sodium salt of the hybridization solution in a concentration wherein it is effective to promote hybridization of the poly(A) nucleic acids to the capture probe.
  • the hybridization composition comprises the sodium salt of the hybridization solution in a concentration ⁇ 250 mM.
  • the hybridization composition may comprise the sodium salt of the hybridization solution in a concentration selected from 25 mM to 250 mM, 35 mM to 200 mM, 40 mM to 175 mM, 50 mM to 150 mM, 55 mM to 125 mM, 60 mM to 115 mM and 60 mM to 100 mM.
  • the hybridization solution used to establish the hybridization conditions in the hybridization composition comprises the sodium salt in a concentration ⁇ 500 mM.
  • the hybridization solution may comprise the sodium salt in a concentration that lies in a range selected from 50 mM to 500 mM, 75 mM to 400 mM, 85 mM to 350 mM, 100 mM to 300 mM, 115 mM to 250 mM, 120 mM to 225 mM and 125 mM to 200 mM.
  • the hybridization solution comprises the sodium salt is a concentration that lies in a range selected from 125 mM to 175 mM.
  • hybridization solution provides particularly good results when being contacted with an equal volume of sample (or diluted sample).
  • hybridization conditions may be established in the hybridization composition as described in the previous paragraph.
  • sodium chloride is preferably used as sodium salt.
  • the hybridization composition comprises the sodium salt of the hybridization solution in a concentration that is ⁇ 200 mM, ⁇ 175 mM, ⁇ 150 mM, ⁇ 125 mM or ⁇ 100 mM.
  • concentrations of the sodium salt in the hybridization composition are preferred, as they provide stringent conditions for specific hybridizing the poly(A) nucleic acids to the capture probe, whereas unspecific hybridization of non-poly(A) nucleic acids is highly diminished.
  • a reduction of the sodium salt concentration in the hybridization solution/hybridization composition had a dramatic effect on non-poly(A) nucleic acid binding.
  • a sodium salt such as sodium chloride in combination with a quaternary ammonium salt therefore provides hybridization conditions that advantageously provide good poly(A) nucleic acid yields while effectively reducing contaminations of the isolated poly(A) nucleic acid with non-poly(A) nucleic acids.
  • the hybridization solution and hence the hybridization composition comprises a quaternary ammonium salt.
  • a quaternary ammonium salt is a tetraalkylammonium salt.
  • the tetraalkylammonium salt may be a tetramethylammonium salt (TMA) or a tetraethylammonium salt (TEA).
  • Suitable tetraalkylammonium salts include but are not limited to tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetraethylammonium nitrate (TEAN), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB).
  • TEAC tetraethylammonium chloride
  • TMAC tetramethylammonium chloride
  • TEAN tetraethylammonium nitrate
  • TMAN tetramethylammonium nitrate
  • TEAB tetraethylammonium bromide
  • TMAB tetramethylammonium bromide
  • the quaternary ammonium salt is not tetramethylammonium sulfate.
  • the quaternary ammonium salt is a tetraalkylammonium salt selected from the group consisting of tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB).
  • TEAC tetraethylammonium chloride
  • TMAC tetramethylammonium chloride
  • TMAN tetramethylammonium nitrate
  • TAB tetraethylammonium bromide
  • TMAB tetramethylammonium bromide
  • tetramethylammonium bromide is used as quaternary ammonium salt.
  • the quaternary ammonium salt is present in a concentration wherein it can support in combination with the sodium salt binding of the poly(A) nucleic acid to the capture probe.
  • the hybridization composition comprises the quaternary ammonium salt of the hybridization solution in a concentration ⁇ 3 M, ⁇ 2.5 M, ⁇ 2 M or ⁇ 1.5 M.
  • the hybridization composition may comprise the quaternary ammonium salt in a concentration ⁇ 100 mM, ⁇ 125 mM, ⁇ 250 mM or ⁇ 375mM.
  • the hybridization composition comprises the quaternary ammonium salt in a concentration selected from 0.1 M to 1.75 M, 0.125 M to 1.5 M, 0.25 M to 1.25 M, 0.375 M to 1 M and 0.375 M to 0.75 M.
  • the quaternary ammonium salt preferably is a tetraalkylammonium salt and suitable examples were described above.
  • the hybridization solution used for establishing the hybridization conditions in the hybridization composition comprises the quaternary ammonium salt in a concentration ⁇ 6 M, ⁇ 5 M, ⁇ 4 M or ⁇ 3 M.
  • the hybridization solution may comprise the quaternary ammonium salt in a concentration ⁇ 200 mM, ⁇ 250 mM, ⁇ 500 mM or ⁇ 750 mM.
  • the hybridization solution comprises the quaternary ammonium salt in a concentration selected from 0.2 M to 3.5 M, 0.25 M to 3M, 0.5 M to 2.5 M, 0.75 M to 2 M and 0.75 M to 1.5 M.
  • such hybridization solution provides particularly good results when being contacted e.g. with an equal volume of sample (or diluted sample).
  • hybridization conditions may be established in the hybridization composition as described in the previous paragraph.
  • respective concentrations of the sodium salt, which preferably is sodium chloride and the quaternary ammonium salt, which preferably is a tetraalkylammonium salt such as tetramethylammonium bromide, in the hybridization composition and/or hybridization solution provide particularly good results. Suitable concentrations can also be determined by the skilled person following the teachings provided herein.
  • hybridization solution and/or the hybridization composition may comprise further components, non-limiting embodiments are described subsequently.
  • the hybridization composition comprises a detergent.
  • a detergent is not required when following the teachings of the present invention. However, it may be used e.g. to support the denaturation of secondary structures in the poly(A) nucleic acids thereby encouraging hybridization to the capture probe to the poly(A) tail.
  • the detergent may be selected from ionic, zwitterionic and non-ionic detergents. Respective detergents and their use in hybridization reactions are well-known to the skilled person.
  • an ionic detergent is used. Ionic detergents comprise anionic and cationic detergents.
  • a hybridization composition comprising an anionic detergent such as SDS or LiDS provides good results.
  • Suitable concentration ranges for the detergent in the hybridization composition include e.g. 0.025% to 5% and may be selected from 0.05% to 3%, 0.75% to 2.5%, 0.1% to 2.25% and 0.15% to 2%.
  • the detergent is preferably comprised in and hence added by the hybridization solution.
  • Suitable concentration ranges for the detergent in the hybridization solution include e.g. 0.05% to 10% and may be selected from 0.1% to 7.5%, 0.25% to 5%, 0.5% to 3% and 0.75% to 2%.
  • lower concentration ranges of 0.1 % to 2%, 0.15% to 1% and 0.2% to 0.5% are suitable.
  • the detergent could, however, also origin from a treatment of the sample depending on the sample type processed. E.g. if a lysate is processed as nucleic acid containing sample and a detergent was used to lyse the sample. However, a detergent is not required. Therefore, according to one embodiment, the hybridisation composition does not contain a detergent.
  • the hybridization composition may comprise a chelating agent.
  • Chelating agents include, but are not limited to ethylenedinitrilotetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA) and furthermore, e.g. citrate or oxalate.
  • EDTA is used as chelating agent.
  • the term "EDTA” indicates inter alia the EDTA portion of an EDTA compound such as, for example, Na 2 EDTA, K 2 EDTA or K 3 EDTA.
  • Suitable concentration ranges for the chelating agent in the hybridization composition include e.g. 0 to 0.25M, 0.5mM to 50mM, 0.75mM to 10mM and 1mM to 5mM.
  • the chelating agent is preferably comprised in and thus added by the hybridization solution.
  • the hybridization solution comprises the chelating agent in a concentration that is selected from the range 0 to 0.5M, 1mM to 100mM, 1.5mM to 20mM and 2mM to 10mM.
  • the chelating agent could, however, also origin from a treatment of the sample depending on the sample type processed.
  • the hybridization solution comprises a buffering agent such as e.g. Tris or other biological buffering agent such as MOPS, HEPES, MES or BIS-TRIS. Suitable examples of buffering agents are also known to the skilled person.
  • a buffering agent such as e.g. Tris or other biological buffering agent such as MOPS, HEPES, MES or BIS-TRIS.
  • Suitable examples of buffering agents are also known to the skilled person.
  • the pH value of the hybridization solution may e.g. lie in a range selected from 6 to 9.
  • the hybridization solution may also comprise water or other solvent suitable to dissolve the components of the hybridization solution.
  • water or another suitable dilution solution can be added separately to the sample to dilute the sample and adjust a certain volume for the (diluted) nucleic acid containing sample that is then to be mixed with a certain volume of the hybridization solution.
  • Such dilution also dilutes the hybridization solution, thereby assisting in establishing the hybridization conditions in the hybridization composition.
  • a respective dilution has the advantage that the hybridization solution can be provided in a concentrated form.
  • variable sample volumes can be easily processed with the same amount of hybridization solution.
  • the given sample volume can be filled up with a dilution solution such as water to a given volume that is then mixed e.g. with an equal (or other predetermined) volume of hybridization solution.
  • other ratios of sample/diluted sample to hybridization can be used depending on the composition of the hybridization solution.
  • the hybridization solution does not comprise chaotropic ions.
  • the hybridization conditions used in the method according to the invention are based on a balanced combination of a sodium salt and a quaternary ammonium salt which result in an efficient isolation of poly(A) nucleic acids while preventing carry-over of non-poly(A) nucleic acids. Therefore, the hybridization solution and respectively the hybridization composition does not contain other hybridization promoting salts besides the sodium salt and the quaternary ammonium salt in a concentration that would counteract these advantageous effects.
  • the hybridization solution and/or hybridization composition does not contain hybridization promoting salts such as lithium or potassium chloride or other non-sodium halides, MgCl 2 and/or chaotropic salts in a concentration that would counteract the advantageous effects achieved by the combination of the sodium salt and the quaternary ammonium salt.
  • the concentration of such salts if at all present is 100mM or less, 75mM or less, 50mM or less or 25mM or less in the hybridization composition and/or hybridization solution.
  • the hybridization solution does not contain any hybridization promoting salts besides the sodium salt and the quaternary ammonium salt.
  • Non-limiting preferred embodiments of the hybridization composition and hybridization solution in particular with respect to the contained sodium salt, which preferably is sodium chloride, and the quaternary ammonium salt, which preferably is a tetraalkylammonium salt, are described in the following.
  • the volume contributed by the capture probe and means used to assist the separation, in particular the solid support (if used for assisting the capturing and separation) is not considered in the determination of the concentration of the described components in the hybridization composition.
  • the components of the hybridization solution can be added as single solution to the sample or may be added separately in any order to the sample, e.g. using two or more solutions comprising at least one chemical of the hybridization solution to generate the hybridization solution.
  • the hybridization composition comprises the sodium salt of the hybridization solution in a concentration ⁇ 250 mM and a tetraalkylammonium salt as quaternary ammonium salt.
  • concentration should be ⁇ 25 mM, preferably ⁇ 35 mM, more preferred ⁇ 50mM.
  • a hybridization solution may be used which comprises a sodium salt in a concentration ⁇ 500 mM and a tetraalkylammonium salt as quaternary ammonium salt.
  • the hybridization solution comprises the sodium salt in a concentration of ⁇ 50 mM, preferably ⁇ 75 mM, more preferred ⁇ 100 mM.
  • the hybridization composition comprises the sodium salt of the hybridization solution in a concentration selected from 25 mM to 175 mM, 50 mM to 150 mM, 55 mM to 125 mM, 60 mM to 115 mM and 60 mM to 100 mM and a tetraalkylammonium salt as quaternary ammonium salt in a concentration selected from 0.25M to 1.25M, 0.375 M to 1 M and 0.375 M to 0.75 M salt.
  • a hybridization solution may be used which comprises a sodium salt in a concentration selected from 50 mM to 350 mM, 100 mM to 300 mM, 115 mM to 250 mM, 120 mM to 225 mM and 125 mM to 200 mM and a tetraalkylammonium salt as quaternary ammonium salt in a concentration selected from from 0.5 M to 2.5 M, 0.75 M to 2 M and 0.75M to 1.5 M.
  • the hybridization composition comprises the sodium salt of the hybridization solution in a concentration selected from 37.5 mM to 125 mM, 50 mM to 100 mM and 55 mM to 87.5 mM and 60 mM to 100 mM and a tetraalkylammonium salt as quaternary ammonium salt in a concentration selected from 0.25M to 1.25M, 0.375 M to 1 M and 0.375 M to 0.75 M, wherein the sodium salt is sodium chloride.
  • a hybridization solution may be used which comprises sodium chloride in a concentration selected from 75 mM to 250 mM, 100 mM to 200 mM and 125 mM to 175 mM and a tetraalkylammonium salt selected from the group consisting of tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB) as quaternary ammonium salt in a concentration selected from from 0.5 M to 2.5 M, 0.75 M to 2 M and 0.75 M to 1.5 M.
  • TEAB tetraethylammonium bromide
  • TMAB tetramethylammonium bromide
  • a detergent may additionally be comprised in the hybridization composition and can be introduced by the hybridization solution. Details are described above and it is referred to the respective disclosure.
  • the hybridization composition furthermore comprises a capture probe capable of hybridizing to the single-stranded poly(A) stretch of the poly(A) nucleic acids.
  • the capture probe may be at least partially complementary to the single-stranded poly(A) stretch of the poly(A) nucleic acids, thereby allowing hybridization of the poly(A) nucleic acids via the poly(A) stretch such as in particular their poly(A) tail to the capture probe.
  • the capture probe will comprise a single-stranded sequence that can hybridize to the poly(A) stretch of the poly(A) nucleic acid. It may also consist of such sequence.
  • a hybridization composition comprising "a" capture probe as used herein also encompasses embodiments wherein the hybridization composition comprises two or more different capture probes.
  • the capture probe is a capture oligonucleotide.
  • the capture oligonucleotide sequence is designed to provide sufficient complementarity to the poly(A) stretch of poly(A) nucleic acids to allow specific hybridization to the poly(A) stretch that is sufficiently strong for the subsequent separation procedure, wherein the captured poly(A) nucleic acid is separated from non-poly(A) nucleic acids and other contaminations.
  • Capture oligonucleotides that are suitable for such purpose are well-known in the art and therefore, do not need any detailed description. Nevertheless, some non-limiting embodiments are described subsequently.
  • the capture probe may comprise or may consist of RNA, DNA, PNA (peptide nucleic acid), LNA (locked nucleic acid) and/or other analogs.
  • analogs of the nucleobase T or U may be used insofar as they allow for hybridization with A residues.
  • Any capture probe that is capable of hybridizing to the poly(A) stretch of poly(A) nucleic acids and hence is e.g. capable of sequence-specific binding to the poly(A) nucleic acid is considered "a capture probe".
  • the capture probe is preferably a capture oligonucleotide such as a synthetic oligonucleotide.
  • the capture oligonucleotide may comprise a poly-pyrimidine sequence capable of hybridizing to the poly(A) stretch such as the poly(A) tail of the poly(A) nucleic acid or a portion thereof, thereby allowing capture of the poly(A) nucleic acid upon hybridization.
  • the capture oligonucleotide comprises at least a stretch of contiguous units such as nucleobases or analogs thereof complementary to the poly(A) stretch, said stretch of the capture oligonucleotide preferably being at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 30, at least 35, at least 40 units long as is well-known in the art.
  • the capture oligonucleotide is at least in the region of complementarity to the poly(A) stretch single-stranded.
  • the whole oligonucleotide may be single-stranded and/or complementary to the poly(A) stretch, respectively to the poly(A) tail.
  • at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of all pairing units e.g.
  • nucleobases of the capture region and/or of the entire capture oligonucleotide are capable of hybridizing to at least a portion of the poly(A) stretch of poly(A) nucleic acids.
  • the capture oligonucleotide hybridizes to the poly(A) stretch of the poly(A) nucleic acid, a double-stranded nucleic acid hybrid is formed, which does not contain any mismatches.
  • the capture oligonucleotide is a poly(T) or poly(U) nucleic acid molecule.
  • a poly(dT) nucleic acid molecule is used.
  • the term a poly(dT) nucleic acid in particular refers to a nucleic acid molecule comprising DNA, that has more than 90% T residues or comprising a DNA region of at least 10, at least 20, at least 25, at least 30 or at least 35 contiguous T residues.
  • Respective capture oligonucleotides are well-known and commonly used for capturing poly(A) nucleic acids such as poly(A) RNA.
  • a poly(dT) nucleic acid is used as capture oligonucleotide for isolating poly(A) RNA, such as in particular poly(A) mRNA, which is a synthetic molecule of which greater than 90% is the nucleobase T.
  • poly(A) mRNA which is a synthetic molecule of which greater than 90% is the nucleobase T.
  • analogs of the nucleobase T or U may be used insofar as they allow for hybridization with A residues.
  • capture oligonucleotides are also commonly referred to as oligo-dT and are well-known in the art.
  • the capture probe may be present in the hybridization composition in free form.
  • the capture probe with the bound poly(A) nucleic acids may then be immobilized to a solid phase during or after the hybridization reaction.
  • the oligonucleotide may also be labeled with a compound that reacts with a second compound that in turn is immobilized to a solid support.
  • the capture probe is provided in an immobilized form wherein the capture probe is attached to a solid support.
  • a solid support functionalized with the capture probe is used and hence is comprised in the hybridization composition. Immobilization to the solid support may be achieved using techniques that are well-known and standard in the art.
  • the oligonucleotide is attached to a solid support using a linker structure.
  • the solid support may be provided by various materials, including but not limited to reaction vessels, microtiter plates, particles, magnetic particles, cellulose, columns, plates, membranes, filter papers and dipsticks or any other solid support that can be used in separation technologies. Any support can be used as long as it allows separation of a liquid phase. Different solid supports were also used in known methods for isolating poly(A) nucleic acids.
  • the solid support is provided by particles commonly also referred to as beads.
  • the particles used may be made of glass, silica, polymers, polystyrene-latex polymers, cellulose and/or plastic.
  • the solid support is provided by a suspension of particles that are functionalized with the capture probe. The use of magnetic particles is preferred.
  • magnetic particles When using magnetic particles as solid support, they may have superparamagnetic, paramagnetic, ferrimagnetic or ferromagnetic properties. Respective magnetic particles can be easily separated by the aid of a magnetic field, e.g. by using a permanent magnet and therefore have advantages with respect to the processing. They are compatible with established robotic systems capable of processing magnetic particles. Here, different robotic systems exist that can be used to process the magnetic particles to which the hybrids of the capture probe and the poly(A) nucleic acid are bound. According to one embodiment, magnetic particles are collected at the bottom or the side of a reaction vessel and the remaining liquid sample is removed from the reaction vessel, leaving behind the collected magnetic particles to which the hybrids are bound. Removal of the remaining sample can occur by decantation or aspiration.
  • the magnet which is usually covered by a cover or envelope, plunges into the reaction vessel to collect the magnetic particles.
  • the sample comprising the magnetic particles can be aspirated into a pipette tip and the magnetic particles can be collected in the pipette tip by applying a magnet e.g. to the side of the pipette tip. The remaining sample can then be released from the pipette tip while the collected magnet particles which carry the bound hybrids remain due to the magnet in the pipette tip. The collected magnetic particles can then be processed further.
  • Such systems are also well-known in the prior art and are also commercially available (e.g. BioRobot EZ1, QIAGEN). Also other processing systems are known and can be used.
  • Particles may also be separated by filtration, centrifugation or by using spin columns that can be e.g. loaded with a suspension of particles as is well-known to the skilled person.
  • the solid support When the solid support is centrifuged it may be pelleted or passed through a centrfugible filter apparatus or column.
  • the capture probe may be biotinylated or otherwise labeled so as to facilitate separation of the hybrids.
  • This embodiment is e.g. suitable when using capture oligonucleotides.
  • Biotin can be derivatized to probe nucleotides, for example using linkers, without impairing the ability of the capture oligonucleotide to hybridize to the poly(A) nucleic acid. Because biotin reacts with avidin/streptavidin, avidin or streptavidin may be employed in conjunction with a biotinylated capture oligonucleotide.
  • the avidin or streptavidin may be linked to a solid support, such as particles or the surface of a vessel where it may bind the biotinylated capture oligonucleotide.
  • the solid support may then be separated from the remainder of the sample e.g. by removing the solid support from the remaining sample or vice versa to isolate the biotinylated capture oligonucleotide, which itself is hybridized to the poly(A) nucleic acid.
  • the capture probes can also be labelled for separation using a number of different modifications that are well known to those of skill in the art.
  • Non-limiting alternatives include labelling the capture probe with an epitope tag and utilizing an antibody or a binding fragment thereof that recognizes that epitope for capture, for example, labelling the oligonucleotides with digoxigenin and using an anti-digoxigenin antibody for capture.
  • haptens may be used for conjugation e.g. with nucleotides or oligonucleotides. Commonly used haptens for subsequent capture include biotin (biotin-11-dUTP), dinitrophenyl (dinitrophenyl-11-dUTP). These modifications include for example fluorescent modifications.
  • fluorescent nucleotide analogs that may be incorporated include but are not limited to Cy3TM-dCTP, Cy3TM-dUTP, CyTM5-dCTP, fluorescein-12-dUTP, AlexaFluor®594-5-dUTP, AlexaFluor®-546-14-dUTP and the like.
  • Fluorescein labels may also be used as a separation moiety using commercially available anti-fluorescein antibodies. Also suitable is the labelling with radioisotopes, enzyme labels and chemiluminescent labels.
  • hybrid binding agents immobilized to a solid support may be used to facilitate separation of the formed hybrids, such as e.g. anti-hybrid binding agents such as anti-DNA/RNA antibodies or binding fragments thereof.
  • anti-hybrid binding agents such as anti-DNA/RNA antibodies or binding fragments thereof.
  • Such embodiments are e.g. suitable in case a RNA/DNA hybrid is formed upon hybridization of the capture probe to the poly(A) nucleic acid.
  • a respective hybrid binding agent could likewise be immobilized to a solid support according to the principles described above.
  • the hybridization composition is incubated under conditions and for a sufficient time to allow hybridization of the poly(A) nucleic acid to the capture probe. As explained, thereby, nucleic acid-hybrids between the poly(A) nucleic acid and the capture probe are formed.
  • the hybridization composition may be gently rocked or otherwise agitated during incubation.
  • the hybridization composition is first heated at a temperature between about 60°C and about 90°C, preferably 65°C to 75°C, prior to incubation under hybridization conditions.
  • a respective heating step assists in disrupting e.g. secondary structures of the poly(A) nucleic acid thereby supporting that the poly(A) stretch such as the poly(A) tail is accessible for hybridization to the capture probe.
  • a respective heating step may be performed for less than 10 min, less than 7 min and preferably less than 5 min.
  • the hybridization composition is incubated at a temperature of 50°C or below, 45°C or below, preferably 40°C or below, more preferred at room temperature, to allow hybridization of the poly(A) RNA to the capture probe.
  • a respective incubation step may be performed for at least 4 min, preferably for a time period that lies in a range of 5 min to 30 min. It is an advantage of the present method that no long incubation times are required and that the hybridization step, including the denaturing step if performed, can be completed in less than 30 min and even less than 20min. However, also longer incubation times can be used if desired.
  • step (a) nucleic acid-hybrids are formed between the poly(A) nucleic acids and the capture probe.
  • step (b) the formed hybrids are separated from the remaining sample. Thereby, the poly(A) nucleic acid is isolated from the sample. The remaining sample is depleted of poly(A) nucleic acids which are bound to the capture probe.
  • separation of the hybrids is preferably assisted by the use of a solid support to which the capture probe and/or the formed hybrids are bound.
  • the solid support with the bound hybrids can be easily separated from the remaining sample.
  • the solid support may be removed from the remaining sample or the remaining sample may be recovered leaving behind the solid support with the bound hybrids.
  • suitable solid supports as well as suitable separation procedures which allow to separate the solid support from the remaining sample are well-known and are also described above in conjunction with the capture probe and it is referred to the respective disclosure which also applies here. Suitable separation procedures are also well-known and available to the skilled person.
  • step (c) the separated hybrids are washed one or more times. Even though this washing step (c) is optional, it is preferably performed in order to support removal of unbound components and impurities that could interfere with certain downstream applications of the isolated poly(A) nucleic acids.
  • one or more washing steps are performed in step (c) in order to further purify the separated poly(A) nucleic acids.
  • This can be conveniently performed e.g. while the hybrids are immobilized to a solid support which preferably is provided by particles.
  • Common washing solutions may be used and suitable embodiments are known to the skilled person.
  • a suitable washing solution removes impurities but substantially does not release the poly(A) nucleic acid from the hybrid to present losses of poly(A) nucleic acids during washing.
  • washing solutions having e.g. the same or a lower concentration of the sodium salt and/or having the same or a lower concentration of the quaternary ammonium salt compared to the used hybridization solution may be used. If more than one washing buffer is used, the salt concentration may be decreased between the washing steps.
  • the particles When particles are used as solid support for immobilizing the capture probe, the particles may be resuspended by agitation, e.g. vortexing during washing.
  • the poly(A) nucleic acids are released from the hybrids. This can be conveniently performed e.g. while the hybrids are immobilized to a solid support which preferably is provided by particles. To achieve release, one or more elution steps may be performed in order to elute the captured poly(A) nucleic acids.
  • any release solution can be used which effects release of the poly(A) nucleic acids from the hybrids and hence allows e.g. to elute the bound poly(A) nucleic acid from the solid support
  • solid support is preferably used to assist the separation of the poly(A) nucleic acids.
  • Respective elution solutions that effectively elute poly(A) nucleic acids from complementary capture probes are known to the skilled person and therefore need no detailed description.
  • Non-limiting examples include water, elution buffers such as TE-buffer and low-salt solutions which have a salt content of 150mM or less, 100mM or less, 75mM or less, 50mM or less, 25mM or less, 20mM or less, 15mM or less, 10mM or less or are salt-free.
  • the elution solution may e.g. comprise a buffering agent, in particular may comprise a biological buffer such as Tris, MOPS, HEPES, MES, BIS-TRIS, propane and others.
  • Elution may be assisted by heating, e.g. to 50°C or above, preferably 60°C or above, more preferred 65°C or above.
  • Elution may also be assisted by shaking what is e.g. particularly feasible if a particulate solid support is used.
  • the method comprises:
  • the method is for isolating poly(A) RNA from a total RNA sample, comprising:
  • the method according to the first aspect allows to efficiently isolate poly(A) nucleic acids from various samples while minimizing a carry-over of non-poly(A) nucleic acids into the isolated poly(A) nucleic acids.
  • nucleic acid or “nucleic acids” as used herein, in particular refers to a polymer comprising ribonucleosides and/or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like.
  • the term encompasses naturally occurring nucleic acids as well as synthetic nucleic acids.
  • poly(A) nucleic acid and corresponding terms in particular refer to a nucleic acid comprising a single stranded poly(A) stretch of consecutive adenine base (“A") residues. Such poly(A) stretch is usually provided at the 3' end of a nucleic acid and then is also referred to as poly(A) tail.
  • poly(A) nucleic acid specifically encompasses any nucleic acid species which comprises a poly(A) tail at its 3' end, in particular sequences having a poly(A) tail of at least 10, at least 15 or at least 20 A residues.
  • poly(A) nucleic acid refers to poly(A) RNA as well as poly(A) DNA.
  • the poly(A) nucleic acid preferably is poly(A) RNA, in particular poly(A) mRNA.
  • Poly(A) RNA is the major polyadenylated nucleic acid of interest for research and diagnostic applications.
  • Non-poly(A) nucleic acids are substantially not captured with the method of the invention due to the advantageous hybridization conditions used.
  • Non-poly(A) nucleic acids i.e. nucleic acids not having a single-stranded poly(A) stretch such as a poly(A) tail, which are not captured and hence are efficiently depleted during the isolation process of the present invention comprise e.g. non-polyadenylated RNA such as rRNA, tRNA, snRNA, snoRNA and also the minor portion of mRNA lacking a poly(A).
  • the term non-poly(A) nucleic acid may also refer to other non-polyadenylated nucleic acids such as non-polyadenylated DNA if present in the sample.
  • nucleic acid containing sample is used herein in a broad sense and is intended to include a variety of sources and compositions that contain nucleic acids.
  • the nucleic acid containing sample may derive from a biological sample but the term also includes other, e.g. artificial samples which comprise poly(A) nucleic acids such as e.g. nucleic acids that were provided in vitro with a poly(A) tail. It in particular refers to compositions comprising purified nucleic acids that were isolated from a biological sample such as total RNA from which the poly(A) nucleic acid is to be isolated using the method of the invention.
  • the nucleic acid containing sample may also be provided by a biological sample, in particular a lysate of a biological sample.
  • poly(A) nucleic acids such as poly(A) RNA can be isolated from a sample lysate.
  • Exemplary biological samples include, but are not limited to, cell samples, environmental samples, samples obtained from a body, in particular body fluid samples, and human, animal or plant tissue samples.
  • Non-limiting examples include, but are not limited to cells, whole blood, blood products, red blood cells, white blood cells, buffy coat, plasma, serum, swabs, urine, sputum, saliva, semen, lymphatic fluid, amniotic fluid, cerebrospinal fluid, peritoneal effusions, pleural effusions, biopsy samples, fluid from cysts, synovial fluid, vitreous humor, aqueous humor, bursa fluid, eye washes, eye aspirates, plasma, serum, pulmonary lavage, lung aspirates, animal, in particular human, or plant tissues, including but not limited to, liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, cell cultures, as well as lysates, extracts, or materials and fractions obtained from the samples described above.
  • the sample is a biological sample derived from a human, animal or plant.
  • the sample may be selected from the group consisting of cells, tissue, tumor cells, and body fluids such as for example blood, blood products such as buffy coat, plasma and serum, urine, liquor, sputum, stool, CSF and sperm, epithelial swabs, biopsies, bone marrow samples and tissue samples, preferably organ tissue samples.
  • the term "sample” also includes processed samples such as preserved, fixed and/or stabilised samples.
  • the nucleic acid containing sample comprised in the hybridization composition is preferably provided by nucleic acids purified from a respective sample such as e.g. total RNA.
  • the nucleic acid containing sample may also be a crude sample comprising the poly(A) nucleic acids in released form and may be provided by a lysate obtained from a respective biological sample.
  • the nucleic acid containing sample is a purified nucleic acid sample.
  • the nucleic acid containing sample is total RNA and poly(A) RNA is isolated from said total RNA using the method of the invention.
  • Total RNA can be isolated from various samples e.g. using any common RNA purification method. Suitable methods are well-known in the prior art and thus, do not need a detailed description here. Suitable methods include but are not limited to the isolation of RNA using phenol/chloroform based methods, the isolation of RNA using chaotropic agents, alcohol and a solid phase such as in particular a silicon containing solid phase (e.g. silica, glass fibers, silicon carbide), alcohol precipitation, precipitation by other organic solvents, polymers or cationic detergents and the like.
  • a silicon containing solid phase e.g. silica, glass fibers, silicon carbide
  • the nucleic acid containing sample from which the poly(A) nucleic acid, preferably poly(A) RNA, is isolated is a processed biological sample such as a lysed sample.
  • the sample may be lysed using any suitable lysis method that is compatible with poly(A) nucleic acid isolation.
  • Various methods for lysing a biological sample are known to the skilled person and also depend on the specific sample type to be processed.
  • the lysis may e.g. be based on or include a chemical or mechanical lysis procedure and non-limiting examples include lysis methods involving lytic agents such as e.g. detergents, proteolytic enzymes or chaotropic salts, heating, ultrasonification, mechanical disruption and the like.
  • a DNA depleted lysate is used as nucleic acid containing sample.
  • DNA may be removed from the lysate e.g. by performing a DNase digest or by selectively isolating and thus removing DNA from the lysate. Suitable methods for selectively binding and thus removing DNA are for example described in EP 0 880 537 and WO 95/21849 , herein incorporated by reference. E.g. if lysing the sample using chaotropic agents such as chaotropic salts in the absence of short chained alcohols such as ethanol or isopropanol, binding conditions can be established that are selective for DNA, in particular if a silicon containing solid phase is used.
  • the bound DNA can be further used, e.g. further processed, e.g. sequenced, and thus may e.g. optionally be washed and eluted from the nucleic acid binding solid phase thereby providing a DNA fraction which is substantially free of RNA.
  • the bound DNA may also be simply discarded if only RNA is of interest.
  • the RNA containing lysate may be cleared prior to isolating the poly(A) RNA therefrom in order to remove e.g. cell debris and other contaminants.
  • the method according to the present invention is particularly suitable for isolating poly(A) RNA from eukaryotic samples.
  • the method efficiently depletes abundant rRNA such as 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA, mitochondrial 12S rRNA and mitochondrial 16S rRNA.
  • abundant rRNA such as 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA, mitochondrial 12S rRNA and mitochondrial 16S rRNA.
  • an efficient depletion of such unwanted RNA is mandatory as otherwise valuable sequencing capacity is wasted.
  • non-poly(A) RNA such as 5S rRNA, 5.8S rRNA and 28S rRNA was depleted by more than 99%.
  • the amount of non-poly(A) RNA is ⁇ 3%, ⁇ 2%, ⁇ 1.5%, preferably ⁇ 1%, ⁇ 0.75%, ⁇ 0.5%, ⁇ 0.25%, ⁇ 0.15%, ⁇ 0.1%, ⁇ 0.05% in the isolated poly(A) RNA.
  • the non-poly(A) RNA to be depleted is in particular rRNA.
  • Typical rRNA that needs to be efficiently depleted is 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA. Furthermore, also 12 mt and 16 mt rRNA can be efficiently depleted with the method of the invention.
  • the method can be used for the preparation of poly(A) nucleic acids for any purpose for which the isolation of poly(A) nucleic acids such as in particular poly(A) RNA is commonly desired.
  • Non-limiting examples include, but are not limited to the isolation of poly(A) RNA from either total RNA or directly from lysates of biological samples such as cells and tissues, for cDNA synthesis, cDNA library construction, amplification based methods such as reverse transcription PCR, subtractive hybridization, the direct isolation of polyadenylated in-vitro transcripts, oligonucleotides or other nucleic acids, in vitro translation, SAGE technology, expression analysis, expression array and expression-chip analysis, microarray analysis, RNAse and S1 nuclease protection, primer extension, RNA northern, dot, and slot blotting, micro injection and furthermore, for sequencing applications, such as in particular NGS applications.
  • poly(A) nucleic acids may also be used in order to selectively remove poly(A) nucleic acids from samples in case the poly(A) nucleic acids are undesired and shall be removed from a sample. In this case, e.g. washing and elution steps to further purify the isolated poly(A) nucleic acid may be obsolete. However, it may also be of interest to analyze the isolated and removed poly(A) nucleic acids separately from non-poly(A) nucleic acids and hence also wash and elute them in this case.
  • the method according to the invention is particularly advantageous because it is highly efficient thereby minimizing losses of poly(A) nucleic acids due to the isolation process, is highly selective for poly(A) nucleic acids thereby reducing unwanted carry-over of non-poly(A) nucleic acids into the isolated poly(A) nucleic acid fraction, and furthermore, requires minimal hands-on time.
  • the advantageous results can be achieved already after one isolation cycle which is a considerable advantage over prior art methods, which often need at least two enrichment cycles in order to provide sufficiently pure poly(A) nucleic acids. Therefore, the method is highly suitable for simultaneous handling of multiple samples also in high throughput applications.
  • the method according to the present invention is particularly suitable for preparing poly(A) RNA for next generation sequencing (NGS) applications such as transcriptome sequencing.
  • NGS next generation sequencing
  • the present invention provides such a method and therefore, makes an important contribution to the art.
  • purified total RNA is preferably used as nucleic acid containing sample material.
  • the poly(A) RNA enriched eluate that is obtained after the isolation process can be used for construction of a sequencing library. This embodiment will also be explained in further detail in conjunction with the method according to the second aspect and it is referred to the respective disclosure.
  • a method for sequencing poly(A) nucleic acids comprising:
  • step (a) the method according to the first aspect is performed in order to isolate poly(A) nucleic acids from a sample. Details of said method and associated advantages are described above and we refer to the respective disclosure which also applies here.
  • poly(A) RNA is isolated as poly(A) nucleic acid.
  • the poly(A) RNA is preferably isolated from a total RNA sample according to step (a) and (b) of the method according to the first aspect and is washed and eluted according to steps (c) and (d) of the method according to the first aspect as is described above.
  • sequencing comprises preparing from the isolated poly(A) nucleic acids a sequencing library.
  • said sequencing library is suitable for massive parallel sequencing and sequencing comprises sequencing in parallel the molecules comprised in the library.
  • Respective sequencing libraries are known in the art.
  • a sequencing library may comprise a plurality of double-stranded molecules and preferably is suitable for massive parallel sequencing and accordingly, is suitable for next generation sequencing. Preparation of a respective sequencing library is also the present standard in transcriptome sequencing.
  • the plurality of double stranded nucleic acid molecules present in the sequencing library may be linear or circular, preferably, the nucleic acid molecules comprised in the sequencing library are linear.
  • a sequencing library which is suitable for next generation sequencing can be prepared using methods known in the prior art.
  • the double-stranded molecules in the sequencing library are DNA molecules.
  • poly(A) RNA may be reverse transcribed to cDNA in case the poly(A) nucleic acid is poly(A) RNA.
  • methods for preparing a sequencing library suitable for next generation sequencing include obtaining DNA fragments optionally followed by DNA repair and end polishing and, finally, often NGS platform-specific adaptor ligation.
  • the obtained cDNA can be fragmented for example by shearing, such as sonification, hydro-shearing, ultrasound, nebulization or enzymatic fragmentation, in order to provide DNA fragments that are suitable for subsequent sequencing.
  • fragmentation to the desired length may occur on the RNA level and thus prior to cDNA synthesis.
  • the isolated poly(A) RNA may be fragmented by magnesium-catalysed hydrolysis of the RNA.
  • the length of the fragments can be chosen based on the sequencing capacity of the next generation sequencing platform that is subsequently used for sequencing.
  • the obtained fragments have a length of 1500bp or less, 1000bp or less, 750bp or less, 600bp or less and preferably 500bp or less as this corresponds to the sequencing capacity of most current next generation sequencing platforms.
  • the fragmented DNA can be repaired afterwards and end polished using methods known in the prior art, thereby providing for example blunt ends or nucleotide overhangs, such as A overhangs.
  • adapters can be ligated at the 5' and/or 3' ends of the DNA fragments, preferably at both ends of the obtained fragments.
  • the specific design of the adapters depends on the next generation sequencing platform to be used and for the purposes of the present invention, basically any adaptors used for preparing sequencing libraries for next generation sequencing can be used.
  • the sequencing library may comprise or consist of randomly fragmented double stranded DNA molecules which are ligated at their 3' and 5' end to adapter sequences.
  • the adaptors provide a known sequence and thus provide a known template for amplification and/or sequencing primers.
  • adaptors double-stranded or partially double-stranded nucleic acids of known sequence can be used.
  • the adapters may have blunt ends, cohesive ends with 3' or 5'overhangs, may be provided by Y shaped adapters or by stem-loop shaped adapters (see e.g. US 2009/0298075 ). Y shaped adapters are e.g. described in (see e.g. US 7,741,463 ).
  • the adapters may also provide an individual index thereby allowing the subsequent pooling of two or more sequencing libraries prior to sequencing. As discussed, sequencing is preferably performed on a next generation sequencing platform. In NGS, sequencing is often performed by repeated cycles of polymerase-mediated nucleotide extensions or, in one common format, by iterative cycles of oligonucleotide ligation.
  • clonal separation of single molecules and subsequent amplification is performed by in vitro template preparation reactions like emulsion PCR (pyrosequencing from Roche 454, semiconductor sequencing from Ion Torrent, SOLiD sequencing by ligation from Life Technologies, sequencing by synthesis from Intelligent Biosystems), bridge amplification on the flow cell (e.g. Solexa/Illumina), isothermal amplification by Wildfire technology (Life Technologies) or rolonies/nanoballs generated by rolling circle amplification (Complete Genomics, Intelligent Biosystems, Polonator).
  • in vitro template preparation reactions like emulsion PCR (pyrosequencing from Roche 454, semiconductor sequencing from Ion Torrent, SOLiD sequencing by ligation from Life Technologies, sequencing by synthesis from Intelligent Biosystems), bridge amplification on the flow cell (e.g. Solexa/Illumina), isothermal amplification by Wildfire technology (Life Technologies) or rolonies/nanoballs generated by rolling circle amplification (Complete Genomics, Intelligent
  • Sequencing technologies like Heliscope (Helicos), SMRT technology ( Pacific Biosciences) or nanopore sequencing (Oxford Nanopore) allow direct sequencing of single molecules without prior clonal amplification.
  • the sequencing can be performed on any of the respective platforms using a sequencing library prepared from the isolated poly(A) RNA. Suitable methods for preparing sequencing libraries and next generation sequencing methods are also described in Metzker, 2011, Voelkerding, 2009, and WO12/003374 .
  • step (a) The advantages of preparing the poly(A) nucleic acid such as preferably poly(A) RNA in step (a) using the method of the first aspect with respect to the sequencing results are described above and it is referred to the respective disclosure.
  • an aqueous hybridization solution comprising:
  • the hybridization solution according to the third aspect can be used in conjunction with and for performing the method according to the first, second and fourth aspect of the invention and in particular it can be used to establish favourable binding conditions for capturing the poly(A) nucleic acid or it may can be used to provide stringent washing conditions that assist in removing non-poly(A) nucleic acids that may have been bound during the capture step, e.g. if the advantageous hybridization conditions according to the invention wherein a reduced concentration of a sodium salt is used in combination with a quaternary ammonium salt such as preferably a tetraalkylammonium salt are not used.
  • hybridization solution in particular suitable and preferred hybridization solution components and hybridization solution component concentrations as well as suitable and preferred mixing ratios with the sample (or diluted sample) are described in detail above in conjunction with the method according to the first aspect of the present invention. It is referred to the above disclosure which also applies here. Non-limiting selected embodiments are again described briefly subsequently.
  • the hybridization solution may comprise the sodium salt in a concentration ⁇ 500 mM.
  • the sodium salt preferably is a sodium halide, more preferably sodium chloride.
  • the hybridization solution may comprise the sodium salt in a concentration that lies in a range selected from 50 mM to 500 mM, 75 mM to 400 mM, 85 mM to 350 mM, 100 mM to 300 mM, 115 mM to 250 mM, 120 mM to 225 mM and 125 mM to 200 mM.
  • the hybridization solution comprises the sodium salt in a concentration that lies in a range selected from 125 mM to 175 mM. As is demonstrated by the examples, such hybridization solution provides particularly good results when being contacted with an equal volume of sample (or diluted sample).
  • the hybridization solution may comprise the quaternary ammonium salt in a concentration ⁇ 6 M, ⁇ 5 M, ⁇ 4 M or ⁇ 3 M.
  • the hybridization solution may comprise the quaternary ammonium salt in a concentration ⁇ 200 mM, ⁇ 250 mM, ⁇ 500 mM or ⁇ 750 mM.
  • the hybridization solution comprises the quaternary ammonium salt in a concentration selected from 0.2 M to 3.5 M, 0.25 M to 3M, 0.5 M to 2.5 M, 0.75 M to 2 M and 0.75 M to 1.5 M.
  • the quaternary ammonium salt preferably is a tetraalkylammonium salt such as a tetramethylammonium salt (TMA) or a tetraethylammonium salt (TEA).
  • Suitable tetraalkylammonium salts include but are not limited to tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetraethylammonium nitrate (TEAN), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB).
  • the quaternary ammonium salt is not tetramethylammonium sulfate.
  • the hybridization solution comprises tetramethylammonium bromide as quaternary ammonium salt.
  • the hybridization solution comprises one or more compounds selected from the group consisting of detergents, chelating agents and buffers. Details are described above in conjunction with the first aspect and it is referred to the above disclosure. According to one embodiment, the hybridization solution does not comprise chaotropic ions.
  • the hybridization conditions used in the method according to the invention are based on a balanced combination of a sodium salt and a quaternary ammonium salt which result in an efficient isolation of poly(A) nucleic acids while preventing carry-over on non-poly(A) nucleic acids. Therefore, the hybridization solution does not contain other hybridization promoting salts besides the sodium salt and the quaternary ammonium salt in a concentration that would counteract these advantageous effects.
  • the hybridization solution does not contain hybridization promoting salts such as lithium or potassium chloride or other non-sodium halides, MgCl 2 and/or chaotropic salts in a concentration that would counteract the advantageous effects achieved by the combination of the sodium salt and the quaternary ammonium salt.
  • the concentration of such salts if at all present in the hybridization solution is 100mM or less, 75mM or less, 50mM or less or 25mM or less.
  • the hybridization solution does not contain any hybridization promoting salts besides the sodium salt and the quaternary ammonium salt.
  • the hybridization solution comprises a sodium salt in a concentration ⁇ 500 mM and a tetraalkylammonium salt as quaternary ammonium salt.
  • the hybridization solution may comprise the sodium salt in a concentration of ⁇ 50 mM, ⁇ 75 mM or ⁇ 100 mM.
  • the hybridization solution may comprise e.g.
  • a sodium salt in a concentration selected from 50 mM to 350 mM, 100 mM to 300 mM, 115 mM to 250 mM, 120 mM to 225 mM and 125 mM to 200 mM and a tetraalkylammonium salt as quaternary ammonium salt in a concentration selected from from 0.5 M to 2.5 M, 0.75 M to 2 M and 0.75M to 1.5 M.
  • the hybridization solution may comprise sodium chloride in a concentration selected from 75 mM to 250 mM, 100 mM to 200 mM and 125 mM to 175 mM and a tetraalkylammonium salt selected from the group consisting of tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB) as quaternary ammonium salt in a concentration selected from from 0.5 M to 2.5 M, 0.75 M to 2 M and 0.75 M to 1.5 M.
  • TEAB tetraethylammonium bromide
  • TMAB tetramethylammonium bromide
  • the hybridization solution according to the third aspect can be advantageously used for preparing a hybridization composition as explained above in conjunction with the method according to the first aspect. It is referred to the above disclosure which also applies here. Furthermore, it can be used to establish stringent washing conditions as will be described in detail below.
  • a kit for isolating poly(A) nucleic acids from a sample, comprising:
  • the hybridization solution according to the third aspect is described above and it is referred to the above disclosure. Details and preferred embodiments with respect to the capture probe are also described in conjunction with the method according to the first aspect and it is referred to the above disclosure which also applies here.
  • the capture probe may be a capture oligonucleotide such as preferably a synthetic oligo(T) - or oligo(U)-comprising nucleic acid molecule or a mixture thereof or any other suitable capture probe that is or can be immobilized to a solid support. Suitable and preferred embodiments of the solid support are also described in conjunction with the method according to the first aspect and it is referred to the above disclosure which also applies here.
  • the capture probe is bound to non-magnetic or magnetic particles.
  • the kit is particularly suitable for use in the method according to the first aspect.
  • the kit may comprise instructions and/or information for use.
  • the kit may comprise instructions and/or information regarding the application of a certain volume of the hybridization solution with a certain volume of the nucleic acid containing sample and/or a dilution solution such as water, to achieve effective concentrations of the sodium salt and the quaternary ammonium salt in the hybridization composition.
  • a dilution solution such as water
  • the advantageous hybridization conditions which employ a reduced concentration of a sodium salt in combination with a quaternary ammonium salt not only provide highly stringent and selective capture conditions for poly(A) nucleic acids, but also provide highly stringent washing conditions. These hybridization conditions therefore, can be advantageously used to remove non-poly(A) nucleic acids during washing steps.
  • a method for isolating poly(A) nucleic acids having a single stranded poly(A) stretch from a nucleic acid containing sample comprising:
  • step (a) poly(A) nucleic acids are hybridized to a capture probe capable of hybridizing to the poly(A) stretch of the poly(A) nucleic acids to form nucleic acid-hybrids.
  • a capture oligonucleotide comprising a sequence complementary to the poly(A) tail may be used.
  • any method and hence hybridization conditions can be used for providing the respective hybrids. Therefore, also prior art methods can be used.
  • the hybridization conditions described above in conjunction with the method according to the first aspect are also used in step (a) of the method according to the fifth aspect. Details with respect to the poly(A) nucleic acids and the capture probe are described in conjunction with the first aspect according to the present invention. The respective disclosure also applies here.
  • step (b) the formed hybrids are separated from the remaining sample.
  • any common separation technology can be used. Exemplary embodiments are described above in conjunction with step (b) of the method according to the first aspect. It is referred thereto as the same disclosure applies here.
  • step (c) the separated hybrids are washed with the hybridization solution according to the third aspect. Details and preferred embodiments of the respective hybridization solution are described above and it is referred to the respective disclosure which also applies here.
  • the components of the hybridization solution can be added a single solution for washing (what is preferred) or may be added separately in any order to the hybrids to generate the hybridization solution used for washing.
  • the hybridization solution may also be diluted with an appropriate dilution solution such as e.g. water or other solvent what is preferred.
  • the same ratios of hybridization solution: dilution solution can be used as are described above in the method according to the first aspect for the ratio hybridization solution: sample (or diluted sample).
  • hybridization conditions are established during washing and hence are used in the washing composition which comprises the hybridization solution and the hybrids, which correspond to the hybridization conditions described above for the hybridization composition of the method according to the first aspect. It is referred to the respective disclosure which also applies here.
  • Non limiting embodiments are desired in the following:
  • the hybridization solution comprises its components in a concentrated form that allows it to be diluted with a dilution solution so as to achieve the proper final concentration in the washing composition that is used to wash the hybrids.
  • the hybridization solution used for washing and hence also the washing composition comprises a sodium salt.
  • a sodium salt This also encompasses the use of a mixture of different sodium salts as "a" sodium salt.
  • the sodium salt promotes binding of the poly(A) nucleic acids to the capture probe thereby reducing the risk that poly(A) nucleic acids are lost during washing. It may be an anorganic or organic sodium salt.
  • the sodium salt is a sodium halide.
  • the sodium salt is not a chaotropic salt.
  • the sodium halide is sodium chloride.
  • the washing composition comprises the sodium salt of the hybridization solution in a concentration ⁇ 250 mM.
  • the washing composition may comprise the sodium salt of the hybridization solution in a concentration selected from 25 mM to 250 mM, 35 mM to 200 mM, 40 mM to 175 mM, 50 mM to 150 mM, 55 mM to 125 mM, 60 mM to 115 mM and 60 mM to 100 mM.
  • the washing composition comprises the sodium salt of the hybridization solution in a concentration that is ⁇ 200 mM, ⁇ 175 mM, ⁇ 150 mM, ⁇ 125 mM or ⁇ 100 mM.
  • concentrations of the sodium salt in the washing composition are preferred, as they provide stringent conditions for specific hybridizing the poly(A) nucleic acids to the capture probe, whereas unspecifically hybridized of non-poly(A) nucleic acids are washed away.
  • the hybridization solution and hence the washing composition comprises a quaternary ammonium salt.
  • a quaternary ammonium salt is a tetraalkylammonium salt.
  • the tetraalkylammonium salt may be a tetramethylammonium salt (TMA) or a tetraethylammonium salt (TEA).
  • Suitable tetraalkylammonium salts include but are not limited to tetraethylammonium chloride (TEAC), tetramethylammonium chloride (TMAC), tetraethylammonium nitrate (TEAN), tetramethylammonium nitrate (TMAN), tetraethylammonium bromide (TEAB) and tetramethylammonium bromide (TMAB).
  • TEAC tetraethylammonium chloride
  • TMAC tetramethylammonium chloride
  • TEAN tetraethylammonium nitrate
  • TMAN tetramethylammonium nitrate
  • TEAB tetraethylammonium bromide
  • TMAB tetramethylammonium bromide
  • the quaternary ammonium salt is not tetramethylammonium sulfate.
  • the washing composition comprises the quaternary ammonium salt of the hybridization solution in a concentration ⁇ 3 M, ⁇ 2.5 M, ⁇ 2 M or ⁇ 1.5 M.
  • the washing composition may comprise the quaternary ammonium salt in a concentration ⁇ 100 mM, ⁇ 125 mM, ⁇ 250 mM or ⁇ 375mM.
  • the washing composition comprises the quaternary ammonium salt in a concentration selected from 0.1 M to 1.75M, 0.125 M to 1.5 M, 0.25 M to 1.25 M, 0.375 M to 1 M and 0.375 M to 0.75 M.
  • the quaternary ammonium salt preferably is a tetraalkylammonium salt and suitable examples are described above.
  • the hybridization solution used for establishing the conditions in the washing composition comprises the quaternary ammonium salt in a concentration ⁇ 6 M, ⁇ 5 M, ⁇ 4 M or ⁇ 3 M.
  • Said hybridization solution may comprise the quaternary ammonium salt in a concentration ⁇ 200 mM, ⁇ 250 mM, ⁇ 500 mM or ⁇ 750 mM. It may comprise the quaternary ammonium salt in a concentration selected from 0.2 M to 3.5 M, 0.25 M to 3M, 0.5 M to 2.5 M, 0.75 M to 2 M and 0.75 M to 1.5 M.
  • Suitable concentrations of the sodium salt and the quaternary ammonium salt can also be determined by the skilled person following the teachings provided herein.
  • Non-limiting preferred embodiments of the washing composition in particular with respect to the contained sodium salt, which preferably is sodium chloride, and the quaternary ammonium salt, which preferably is a tetraalkylammonium salt, are described in the following.
  • the volume contributed by the capture probe and means used to assist the separation such as the solid support (if used for assisting the capturing and separation) is not considered in the determination of the concentration of the described components in the washing composition.
  • the components of the hybridization solution can be added as single solution to the sample or may be added separately in any order to the separated hybrids, e.g. using two or more solutions comprising at least one chemical of the hybridization solution to generate the hybridization solution that is used for washing.
  • the washing composition comprises the sodium salt of the hybridization solution in a concentration ⁇ 250 mM and a tetraalkylammonium salt as quaternary ammonium salt.
  • the washing composition comprises the sodium salt of the hybridization solution in a concentration selected from 25 mM to 175 mM, 50 mM to 150 mM, 55 mM to 125 mM. 60 mM to 115 mM and 60 mM to 100 mM and a tetraalkylammonium salt as quaternary ammonium salt in a concentration selected from 0.25M to 1.25M, 0.375 M to 1 M and 0.375 M to 0.75 M salt.
  • the washing composition comprises the sodium salt of the hybridization solution in a concentration selected from 37.5 mM to 125 mM, 50 mM to 100 mM and 55 mM to 87.5 mM and 60 mM to 100 mM and a tetraalkylammonium salt as quaternary ammonium salt in a concentration selected from 0.25M to 1.25M, 0.375 M to 1 M and 0.375 M to 0.75 M, wherein the sodium salt is sodium chloride.
  • the hybridization conditions according to the invention that can also be used to establish stringent washing conditions are based on a balanced combination of a sodium salt and a quaternary ammonium salt which result in an efficient isolation of poly(A) nucleic acids while preventing carry-over on non-poly(A) nucleic acids. Therefore, the hybridization solution and respectively the washing composition does not contain other hybridization promoting salts besides the sodium salt and the quaternary ammonium salt in a concentration that would counteract these advantageous effects.
  • the hybridization solution and/or washing composition does not contain hybridization promoting salts such as lithium or potassium chloride or other non-sodium halides, MgCl 2 and/or chaotropic salts in a concentration that would counteract the advantageous effects achieved by the combination of the sodium salt and the quaternary ammonium salt.
  • the concentration of such salts if at all present is 100mM or less, 75mM or less, 50mM or less or 25mM or less.
  • the hybridization solution does not contain any hybridization promoting salts besides the sodium salt and the quaternary ammonium salt.
  • step (d) poly(A) nucleic acids are released from the washed hybrids. Details with respect to release step (d) have also been described above in conjunction with the method according to the first aspect. It is referred to the above disclosure, which also applies here.
  • washing conditions of the present disclosure at least in one washing step of a poly(A) nucleic acid isolation procedure has the advantage that non- poly(A) nucleic acids that may have bound in the hybridization step (a) (e.g. if the hybridization conditions of the present invention have not been used) can be subsequently washed away.
  • One or more further washing steps can be performed in addition. This is also preferred in order to improve the purity of the poly(A) nucleic acids and to remove components of the hybridization solution that may potentially interfere with downstream applications.
  • the poly(A) nucleic acid preferably is poly(A) RNA. It is referred to the above disclosure made in conjunction with the method according to the first aspect which also applies here.
  • solution refers to a liquid composition, preferably an aqueous composition. It may be a homogenous mixture of only one phase but it is also within the scope of the present invention that a solution comprises solid constituents such as e.g. precipitates.
  • subject matter described herein as comprising certain steps in the case of methods or as comprising certain ingredients in the case of compositions, solutions and/or buffers refers to subject matter consisting of the respective steps or ingredients. It is preferred to select and combine preferred embodiments described herein and the specific subject-matter arising from a respective combination of preferred embodiments also belongs to the present disclosure.
  • RNA isolated from Jurkat cells was used as nucleic acid containing sample from which poly(A) RNA was isolated.
  • capture probe dC 10 T 30 capture oligonucleotides covalently linked to the surface of (non-magnetic) polystyrene-latex particles (Oligotex suspension; QIAGEN) or dT 14 capture oligonucleotides covalently linked to the surface of magnetic particles (Seradyn Magnetic Beads) were used.
  • the performed poly(A) nucleic acid isolation protocol was based on the Oligotex® handbook protocol (Qiagen).
  • the main steps of the Basic Protocol are summarized in the following:
  • Magnetic Protocol Magnetic Protocol
  • total RNA no dilution
  • 50 ⁇ l magnetic particles see above. Hybridization occurred at room temperature for 5min.
  • the particles were washed several times and the poly(A) nucleic acids were eluted with a low salt buffer.
  • RNA detection was performed using SYBR Green reporter dye based quantitative real time RT-PCR assays. The following target RNAs were detected: 18S rRNA (non-poly(A) contamination), 28S rRNA (non-poly(A) contamination) and also other rRNAs (non-poly(A) contamination) and RPL (60S ribosomal protein L12 gene) mRNA, PPIA (peptidylprolyl isomerase A gene) mRNA, CDH2 (cadherin-2 gene) mRNA and/or GAPDH (glyceraldehyde 3-phosphate dehydrogenase gene) mRNA.
  • SYBR Green reporter dye based quantitative real time RT-PCR assays.
  • the following target RNAs were detected: 18S rRNA (non-poly(A) contamination), 28S rRNA (non-poly(A) contamination) and also other rRNAs (non-poly(A) contamination) and RPL (
  • Example 1 demonstrates that the nature of the salt used for hybridization is important.
  • the performed hybridization studies with LiCl, MgCl 2 and KCI containing hybridization solutions showed only a minor or no influence on non-poly(A) recovery (as determined by analysis of rRNA content in the eluates) if these salts were used in different concentrations. Different settings were tested:
  • RNA from Jurkat cells served as starting material for the poly(A) RNA isolation procedure which was performed according to the Magnetic Protocol.
  • the tested hybridization solutions contained a Tris-buffer (pH 7.5), an anionic detergent (LiDs) and LiCl in different concentrations (300 mM, 500 mM or 700 mM).
  • washing buffer 1 150mM LiCl, LiDs
  • 2 150mM LiCl
  • poly(A) RNA isolation was performed manually as follows:
  • the obtained eluates were diluted 1:280 (2 ⁇ l eluate + 558 ⁇ l H2O).
  • Initial total RNA (680ng/ ⁇ l) was diluted 1:1838,27 to receive a concentration of 0.37 ng/ ⁇ l.
  • 5 ⁇ l of diluted pol(A) RNA eluate or diluted total RNA was used in the real time RT-PCR analysis. All approaches were analyzed in duplicates per condition.
  • the mean Ct value was calculated from two parallel repetitions per approach.
  • the Ct value abbreviated for cycle threshold, corresponds to that PCR cycle number at which the fluorescence of the SYBR Green reporter dye first rises exponentially above the background value (threshold).
  • the lower the Ct value the more cDNA template and thus the more initial transcript was present in the eluate.
  • An increase in the Ct value of a transcript indicates that some transcript was lost during the poly(A) RNA enrichment.
  • Table 1 shows the results.
  • Table 1 The mean Ct values of the 18S rRNA and the RPL mRNA as determined by real time RT-PCR analyses are shown as obtained using decreasing concentrations of LiCl in the hybridization solution.
  • the hybridization solution comprised besides water and KCI or MgCl 2 in different concentrations (for each salt: 1 M, 500 mM or 100 mM), 20mM Tris, 2% SDS and 2.5mM EDTA; the pH value was adjusted to 7.5 with HCl or NaOH.
  • a corresponding hybridization solution comprising 1 M NaCl instead of KCI or MgCl 2 was tested as reference standard.
  • RNA purified from Jurkat cells served as starting material.
  • the Basic Protocol as described above under Materials and Methods was followed using as capture oligonucleotide functionalized solid support an Oligotex suspension and spin columns to assist separation.
  • the mean Ct value was determined and the results are shown in Table 2.
  • Table 2 The mean Ct values of the 18S rRNA and of the PPIA and CDH2 mRNA as determined by real time RT-PCR analyses is shown as obtained using decreasing concentrations of KCI or MgCl 2 or 1 M NaCl in the hybridization solution.
  • the effects on rRNA depletion (based on 18S and 28S rRNA) and poly(A) mRNA enrichment (based on GPDH and PPIA mRNA) was analysed when decreasing the concentration of NaCl in the hybridization solution.
  • the hybridization solution comprised besides water and NaCl in different concentrations (1 M, 400 mM, 300 mM, 200 mM, 100 mM or 50mM), 20mM Tris (pH 7.5), 2% SDS and 2.5mM EDTA (pH 8).
  • RNA isolation For poly(A) RNA isolation, the Basic Protocol as described above under Materials and Methods was followed using as capture oligonucleotide functionalized solid support an Oligotex suspension and spin columns to assist separation. 5 ⁇ g total RNA purified from Jurkat cells served as starting material.
  • RNA eluates were diluted 1:1000.
  • Initial total RNA (1.27 ⁇ g/ ⁇ l) was diluted by adding 3.94 ⁇ l RNA to 46.06 ⁇ l RNase free water.
  • 5 ⁇ l of diluted poly(A) RNA eluate or diluted total RNA was used in the real time RT- PCR analysis.
  • Figure 1 shows the delta Ct values of the tested conditions (delta Ct value calculation: mean Ct value determined after poly(A) RNA enrichment minus the mean CT value determined for the initial total RNA sample). All tested hybridization solutions depleted 18S rRNA during poly(A) enrichment, as can be seen from the significantly increased mean Ct value compared to the mean Ct value obtained with the initial total RNA sample.
  • Table 3 The mean Ct values of the 18S rRNA, 28S rRNA and of the GAPDH and PPIA mRNA as determined by real time RT-PCR analyses is shown as obtained using decreasing concentrations of NaCl in the hybridization solution.
  • Example 3 the effect on rRNA depletion and poly(A) mRNA enrichment utilizing a low ionic strength NaCl salt concentration (150 mM) in combination with different quaternary ammonium salts in the hybridization solution was analyzed.
  • the tested hybridization solutions had the following composition: 20mM Tris, 150mM NaCl, 0.2% SDS, EDTA, water and 1M of the tested quaternary ammonium salt.
  • the tetraalkylammonium salts tetramethylammonium chloride (TMAC), tetramethylammonium nitrate (TMA nitrate), tetramethylammonium bromide (TMA bromide) or tetraethylammonium bromide (TEA bromide) were tested.
  • TMAC tetramethylammonium chloride
  • TMA nitrate tetramethylammonium nitrate
  • TMA bromide tetramethylammonium bromide
  • TEA bromide tetraethylammonium bromide
  • RNA (0.5 ⁇ g and 2 ⁇ g) from Jurkart cells served as starting material. The rRNA depletion efficiency was tested based on 18S rRNA and 28S rRNA levels and poly(A) RNA enrichment based on PPIA and GAPDH transcripts.
  • RNA eluates were diluted 1:200.
  • Initial total RNA (785.5ng/ ⁇ l) was diluted by adding 6.37 ⁇ l to 43.63 ⁇ l RNase free water. From this initial dilution a 1:200 dilution was obtained. 5 ⁇ l of diluted poly(A) RNA eluate or diluted total RNA was used in the real time RT- PCR analysis.
  • the mean Ct value for the 18S rRNA and 28S rRNA and GAPDH mRNA and PPIA mRNA per condition using 0.5 ⁇ g or 2 ⁇ g total RNA as starting material was calculated from two parallel experiments per condition.
  • Figure 2 a) to d) shows the delta Ct values (calculated as explained above) obtained with the tested conditions.
  • the effect on rRNA depletion and mRNA recovery is quite comparable when using 0.5 ⁇ g or 2.0 ⁇ g total RNA as starting material for poly(A) RNA isolation.
  • All tested hybridization solutions comprising 150 mM NaCl in combination with a quaternary ammonium salt achieved significantly higher 18S rRNA ( Figure 2 a) and 28S rRNA ( Figure 2 b) depletion rates compared to the 1 M NaCl reference hybridization solution.
  • the hybridization solutions wherein 150mM NaCl was used in combination with a tetraalkylammonium salt showed significantly less unwanted losses in the poly(A) RNA compared to the 150 mM NaCl containing hybridization solution as determined based on the target mRNAs PPIA and GAPDH. Therefore, mRNA losses could be significantly reduced compared to the hybridization solution not containing a tetraalkylammonium salt.
  • the hybridization conditions taught by the present invention wherein a lower concentration of a sodium salt such as sodium chloride is used in combination with a quaternary ammonium salt provide an advantageous balance between non-poly(A) depletion and poly(A) RNA recovery.
  • the tested hybridization solutions had the following composition: 20mM Tris, 150mM NaCl, 0.2% SDS and EDTA, water and 0.5 M, 1 M, 1.5 M or 2 M TMAB.
  • corresponding hybridization solutions comprising 1 M or 150 mM NaCl but no quaternary ammonium salt were tested in parallel.
  • the poly(A) RNA isolation procedure was performed according to the Basic Protocol described above under Materials and Methods using as capture oligonucleotide functionalized solid support Seradyn Magnetic Beads. 2 ⁇ g total RNA isolated from Jurkart cells served as starting material. The rRNA depletion efficiency was tested based on 18S rRNA and 28S rRNA levels and poly(A) RNA enrichment based on PPIA and GAPDH mRNA.
  • RNA eluates were diluted 1:200.
  • Initial total RNA (0.97 ⁇ g/ ⁇ l) was diluted by adding 2.06 ⁇ l to 47.95 ⁇ l RNase free water. From this initial dilution a 1:200 dilution was obtained. 5 ⁇ l of diluted poly(A) RNA eluate or diluted total RNA was used in the real time RT- PCR analysis.
  • Example 4 thereby clearly demonstrates the benefits of the hybridization solution and hybridization composition according to the invention and shows that the tetraalkylammonium salt can be used in different concentrations. These results were also confirmed in other experiments wherein corresponding hybridization solutions comprising 0.2 M TMAB were tested. Thus, the quaternary ammonium salt works in high and low concentrations.
  • the poly(A) RNA isolation procedure was performed according to the Basic Protocol described above under Materials and Methods using as capture oligonucleotide functionalized solid support Seradyn Magnetic Beads.
  • the hybridization solution comprised 20mM Tris, 150mM NaCl, 0.2% SDS water and 1 M TMAB. 5 ⁇ g total RNA isolated from Jurkart cells served as starting material.
  • the residual amount of different rRNAs (5S rRNA, 5.8S rRNA, 12s rRNA, 16S rRNA, 18S rRNA and 28S rRNA) was determined in the isolated poly(A) RNA eluate based on real time RT-PCR analysis. 7 samples were evaluated. The obtained eluates were diluted 1:1000.
  • RNA was diluted to 5.1 ⁇ g/50 ⁇ l. From this initial dilution a 1:1000 dilution was obtained. Further 1:10 dilutions were then prepared. 5 ⁇ l of diluted poly(A) RNA eluate or diluted total RNA was used in the real time RT- PCR analysis.
  • Table 4 residual rRNA in the eluates after poly(A) RNA isolation 5S rRNA Residual rRNA Poly(A) RNA eluate 0.00% Control 100% 5,8S rRNA Residual rRNA Poly(A) RNA eluate 0.00% Control 100% 12S rRNA Residual rRNA Poly(A) RNA eluate 0.22% Control 100% 16S rRNA Residual rRNA Poly(A) RNA eluate 0.91% Poly(A) RNA eluate 1.27% Control 100% 18S rRNA Residual rRNA Poly(A) RNA eluate 0.00% Control 100% 28s rRNA Residual rRNA Poly(A) RNA eluate 0.00% Control 100%
  • DMSO dimethyl methacrylate
  • TMAC trimethyl methacrylate
  • betaine a substance added to a hybridization solution containing 500mM LiCl
  • 5 ⁇ g total RNA isolated from Jurkart cells served as starting material for poly(A) nucleic acid enrichment using the Magnetic Protocol described above.
  • the hybridization composition was prepared by mixing 4.20 ⁇ l total RNA, 370 ⁇ l hybridization solution and 50 ⁇ l magnetic particles functionalized with the capture oligonucleotide.
  • LiCl buffer (Tris pH: 7.5, 500mM LiCl, 1% anionic detergent)
  • LiCI - DMSO composition as reference LiCl buffer + DMSO (final: 5%)
  • LiCI - Formamide composition as reference LiCl buffer + formamide (final: 2%)
  • LiCI - TMAC composition as reference LiCl buffer + TMAC (final: 100 nM)
  • LiCI - Betaine composition as reference LiCl buffer + betaine (final: 1 M)
  • Table 5 shows the result of the quantitative real time RT-PCR analysis.
  • the delta CT values were determined for 18S and 28S rRNA to analyse non-poly(A) depletion and GAPDH and CDH2 to analyse mRNA enrichment.
  • Table 5 Delta Ct values of the 18S rRNA and 28S rRNA and of the GAPDH and CDH2 mRNA are shown.
  • the reference hybridization buffer (see above) was supplemented with different concentrations of TMAC (see table 6 below) to analyse whether higher TMAC concentrations could improve the results.
  • TMAC concentrations of TMAC
  • Table 6 Delta Ct values of the 18S rRNA and 28S rRNA and of the GAPDH and CDH2 mRNA are shown.

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EP14166712.1A 2014-04-30 2014-04-30 Method for isolating poly(A) nucleic acids Withdrawn EP2940136A1 (en)

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EP14166712.1A EP2940136A1 (en) 2014-04-30 2014-04-30 Method for isolating poly(A) nucleic acids
CN201580023578.1A CN106459965A (zh) 2014-04-30 2015-04-28 分离多聚(a)核酸的方法
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PCT/EP2015/059117 WO2015165859A1 (en) 2014-04-30 2015-04-28 Method for isolating poly(a) nucleic acids
US15/129,280 US20180179514A1 (en) 2014-04-30 2015-04-28 Method for isolating poly(a) nucleic acids
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